The long-term goal of this work is to understand how epigetically heritable silent chromatin domains are assembled and inherited. Silent domains are a conserved feature of eukaryotic chromosmes and play important roles in regulation of gene expression and maintenance of chromosome stability. The budding yeast Saccharomyces cerevisiae contains an example of epigenetically heritable silent chromatin domains that are amenable to both genetic and biochemical analysis. We have purified the silencing complexes that act at different chromosome regions in budding yeast and have investigated their activities and mechanism of localization to chromatin. In the SIR (Silent Information Regulator) complex, which mediates telomeric and mating-type silencing, the NAD-dependent deacetylase Sir2 associates with histone binding proteins Sir3 and Sir4, and all three proteins spread along the chromatin fiber in a deacetylation-dependent manner. In the RENT (Regulator of Nucleolar silencing and Telophase) complex, which inhibits inappropriate recombination and transcription from foreign promoters within the ribosomal DMA (rDNA) repeats, Sir2 associates with Net1 and Cdc14, as well as a putative rDNA sister chromatid clamp complex (termed rSC, rDNA Sister chromatid Clamp). In this proposal, we will investigate how the SIR complex deacetylates and binds to its chromatin target and how deacetylation and other interactions drive SIR complex assembly and spreading along the chromatin fiber. In addition, we will investigate how the newly identified rSC complex regulate rDNA structure. These studies are aimed at a complete molecular understanding of gene silencing through the molecular dissection of in vitro reconstituted silent chromatin. The conservation of silent chromatin domains and Sir2-like deacetylases suggests that the principles developed here for the budding yeast complexes will apply in other settings. A basic understanding of the mechanism of gene silencing will not only provide a frame work for understanding how the process can fail, but also provides the substrate and knowledge to design therapeutic strategies based on intervention.